Androgen Activity: A clinical need to assay the function of the androgen receptor (AR) occurs when defects appear in the pathway of androgen action. For example, mutations in the AR affect the bioactivity of the receptor in Androgen Insensitivity Syndrome, AIS, (Kazemi-Esfarjani et al., 1993; Brinkmann et al., 1991; Brinkmann et al., 1992a; Brinkmann et al., 1992b; De Bellis et al., 1992; French et al., 1990; Imperato-McGinley et al., 1990; Lubahn et al., 1989; Quigley et al., 1992; Ris-Stalpers et al., 1990; Ris-Stalpers et al., 1991; Simental et al., 1992) or testicular feminized animals (Yarbrough et al., 1990; He et al., 1991), Kennedy Syndrome (La Spada et al., 1991), prostate cancer (Newmark et al., 1992; Brinkmann et al., 1991; Veldscholte et al., 1992b; Veldscholte et al., 1992a; Veldscholte et al., 1990) and breast cancer (Wooster et al., 1992). Besides mutations directly in the receptor, defects can occur in the non-androgenic mechanism for steroid receptor activation as has been reported for steroid receptors (Power et al., 1991; Shemshedini et al., 1992; Kuiper et al., 1993). An assay that would measure the extent of these defects would also provide a tool to test new materials that may activate the defective receptor and form the basis of a therapy.
Androgen receptors are members of a nuclear receptor superfamily which are believed to function primarily as transcription factors that regulate gene activity through binding specific DNA sequences to hormone responsive elements (HRE) and associated factors (Allan et al., 1991; Smith et al., 1993; Evans, 1988; Beato, 1989). In general, these HREs can be grouped into two categories of inverted repeat consensus sequences: the TGACC motif that mediates estrogen, retinoic acid, and thyroid hormone responses (Klein-Hitpass et al., 1986; Umesono et al., 1988); and the TGTTCT sequence that confers regulation by glucocorticoids, progestins and androgens (Scheidereit et al., 1986; Shrahle et al., 1987; Ham et al., 1988). The inclusion of the androgen receptor responsive element (ARE) in this latter group is based largely on observed binding of androgen receptors to the glucocorticoid responsive element (GRE) of mouse mammary tumor virus (MMTV) DNA (Ham et al., 1988; Roche et al., 1992; Darbre et al., 1986; Cato et al., 1987) and the tyrosine aminotransferase (TAT) gene (Denison et al., 1989).
Androgen regulation of the C3 (1) gene which encodes a polypeptide component of prostatic steroid-binding protein has been investigated (Heyns et al., 1978; Hurst et al., 1983; Parker et al., 1988; Parker et al., 1980). Although sequences within both the promoter region and first intron of the C3 (1) gene have high affinity binding for androgen receptors (Perry et al., 1985; Claessens et al., 1993; De Vos et al., 1991; Rushmere et al., 1990), attempts to use these sequences to confer androgen regulation on a homologous or heterologous promoter-reporter system have met with limited success (Parker et al., 1985; Parker et al., 1988); with only a weak androgen induction seen with these genomic fragments (Claessens et al., 1993; Tan et al., 1992; De Vos et al., 1991; Rushmere et al., 1990; Claessens et al., 1990b; Claessens et al., 1990a; Claessens et al., 1989b; Claessens et al., 1989a). Recently, DNase I footprinting experiments have shown that the DNA-binding domain of the androgen receptor binds to a glucocorticoid responsive element (GRE) present in this intronic fragment (De Vos et al., 1991; Claessens et al., 1993). The occurrence of a complete GRE in this gene is consistent with the observed effects of glucocorticoids on the expression of the C1 component of prostatic binding protein (Rennie et al., 1989). The human prostate specific antigen (PSA) gene is androgen regulated in human prostate tumors and in cell culture (Riegman et al., 1991; Montgomery et al., 1992; Young et al., 1992; Murphy et al., 1992; Armbruster, 1993). Construction of the PSA DNA promoter reveal a GRE-like sequence that responds to androgens (Riegman et al., 1991). The Slp gene demonstrates specific androgen regulation via GRE-like sequences (Adler et al., 1992; Adler et al., 1991). Other androgen regulated genes from the prostate have been cloned, such as the SVS II (Dodd et al., 1983; Dodd et al., 1986; Harris et al., 1990), 20 kDa protein (Ho et al., 1989), and DP1 (Ho et al., 1992), but the androgen regulatory sequences have not been identified.
Transcenic animals: The introduction of a gene into the germline at the one cell or early embryonic stage produces a transgenic animal which will contain and pass on the gene to its offspring. Tissue specific expression of a gene can be restricted by tissue specific elements with the DNA. Success with prostate specific expression of transgenes has been limited and often not restricted to the prostate. For example, the complete rat C3(1) gene including 4.3 kb of 5'-flanking sequence and 2.2 kb of 3'-flanking sequence will give prostate specific expression in transgenic mice (Allison et al., 1989), but using only the 6 kb of 5'-flanking C3(1) resulted in transgenic lines that targeted to the prostate, seminal vesicles, and testis (Buttyan et al., 1993). MMTV coupled to int-2 produced a transgenic mouse line that developed prostatic epithelial cell hyperplasia that was androgen regulated but the males are sterile (Muller et al., 1990; Leder, 1990; Tutrone et al., 1993). Various males in different lines, in addition to expressing the transgene in the prostate, also expressed the int-2 gene in the seminal vesicles, vas deferens, salivary gland while the females expressed the gene in the mammary gland and developed mammary hyperplasia (Leder, 1990). However, targeting with MMTV can lead to expression in the testis resulting in sterility (Lucchini et al., 1992). Using the gp91-phox gene promoter (a gene not normally expressed in the prostate) linked to the early region of SV-40 virus, lesions in the prostate defined as neuroblastomas were created (Skalnik et al., 1991) .
Any gene targeted to the prostate in transgenic animals may alter prostatic growth and function. Oncogenes and tumor suppressor genes (Fleming et al., 1986; Matusik et al., 1987; Dodd et al., 1990; Hockenbery, 1992; Buttyan et al., 1993; Carter et al., 1990a; Carter et al., :1990b; Tutrone et al., 1993; Bookstein et al., 1993; Thompson et al., 1993; Peehl, 1993; Dodd et al., 1993; McNicol et al., 1991; McNicol et al., 1990a; McNicol et al., 1990b) as well as growth factors (Morris et al., 1990; Ichikawa et al., 1992; Isaacs et al., 1991a; Isaacs et al., 1991b; Carter et al.,1990a; Pienta et al., 1991; Morton et al., 1990) implicated in the development of prostatic hyperplasia or cancer are likely starting points. In addition, genes such as the large T antigen, which successfully induce cancer in endocrine glands when targeted in transgenic animals, are suitable candidates (Anonymous, 1991; Stefaneanu et al., 1992; Hanahan, 1986; Rindi et al., 1991; Hamaguchi et al., 1990).
Transgenic animals that express the transgene in a tissue or non-tissue specific can result in new models. For example, non-tissue specific expression can result in diseased states in a number of tissues while tissue specific expression of targeted genes can lead to disease states in targeted organs as follows: cancer models (Burck et al., 1988; Yamamura, 1989; Folkman et al., 1989; Reynolds et al., 1988; Anonymous, 1992; Bautch, 1989; Hanahan, 1986; Lucchini et al., 1992; Anonymous, 1988); mammary adenocarcinoma (Muller et al., 1988; Muller, 1991; Pawson, 1987; Callahan et al., 1989; Muller, 1991; Strange et al., 1990); hyperplasia and dysplasia (Mayo et al., 1988; Borrelli et al., 1992; Eva et al., 1991; Lin et al., 1992; Matsui et al., 1990); neuroblastomas (Dalemans et al., 1990); liver cancer (Butel et al., 1990; Dubois et al., 1991; Sandgren et al., 1993; Sandgren et al., 1989); gonadal tumors (Schechter et al., 1992; Matzuk et al., 1992); thymic mesenchymal tumors (Sinkovics, 1991); and leukaemia (Knight et al., 1988; Adams et al., 1985). Further, targeted genes may function to accelerate tumor formation by conferring susceptibility to transformation by factors or carcinogens (Langdon et al., 1989; Breuer et al., 1989; Mougneau et al., 1989). Promoters, such as metallothionein (MT), often lead to general expression in many organs (Dyer et al., 1989; Iwamoto et al., 1991) while the MMTV promoter limits expression to endocrine target tissues due to its HRE (Ham et al., 1988; Roche et al., 1992; Darbre et al., 1986; Cato et al., 1987). Even using a general promoter can lead to specific effects if the factor expressed targets a specific tissue, i.e. MT-growth hormone releasing factor (Mayo et al., 1988) or ectopic nerve growth factor (Borrelli et al., 1992) lead to pituitary hyperplasia in transgenic mice.
Gene therapy: The treatment of human disease or disease in non-human eukaryotic animals by gene therapy started with the goal to correct single-gene inherited defects. Advances have expanded that goal to include the treatment of acquired diseases, such as cancers (Davies, 1993; Anderson, 1992; Mulligan, 1993; Culotta, 1993; Felgner, 1993; Tolstoshev et al., 1993). Approved clinical trials are presenting encouraging results. The practical problem has been the development of efficient and specific approaches that will transfer and express a gene within the correct cell type. The approaches can be classed as viral and nonviral methods to transfer genes. Some of the therapeutic approaches transfer the gene(s) to patient cells which have been cultured and then returned to the same individual (Fenjves et al., 1989). Others attempt direct transfer of the gene to the human tissue. For example, a DNA complex with liposomes can be delivered to the airway and correct the cystic fibrosis defect in transgenic mice (Hyde et al., 1993). Direct gene transfer by DNA: cationic liposomes into adult mice demonstrates efficient transfer and expression occurs in most organs (Zhu et al., 1993). If the gene is stably integrated, then the defect may be corrected while, if the gene was transiently expressed, then a relief in the disease would likely be transient. However, in cases, such as cancer, where the goal is to kill the cancerous cell, transient expression would be sufficient if the expressed gene is toxic (Short et al., 1990; Culver et al., 1992). Approaches may include expressing tumor suppressor genes (Friedmann, 1992) or genes to inhibit expressed oncogenes (Mukhopadhyay et al., 1991).